CN1500148A - Methods for prodn. of products in host cells - Google Patents

Abstract

The invention provides methods and host cells for the production of ascorbic acid intermediates. The invention also provides host cells having a modification in a polynucleotide that uncouples the catabolic pathway from the oxidative pathway by deleting the encoding for an endogenous enzymatic activity that phosphorylates D-glucose at its 6th carbon and/or a polynucleotide that has deleted the encoding for endogenous enzymatic activity that phosphorylates D-gluconate at its 6th carbon. Such host cells are used for the production of products, such as, ascorbic acid intermediates. Nucleic acid and amino acid sequences with inactivated enzymatic activity which phosphorylates D-glucose at its 6th carbon and inactivated enzymatic activity which phosphorylates D-gluconate at its 6th carbon are provided.

Description

Method for producing products in host cells

Statement of authority to accomplish invention under federally sponsored research

The invention was made with U.S. government support under fund No.70 NANB 5H1138 funded by the U.S. department of commerce. The government has certain rights in the invention.

Technical Field

The present invention relates to engineering metabolic pathways of host cells and provides methods and systems for producing products in host cells. The present invention provides, inter alia, methods and systems for producing ascorbic acid intermediates in host cells.

Background

Biocatalytic production of many products of commercial interest, such as L-ascorbic acid intermediates, has been utilized in genetically engineered host cells. L-ascorbic acid (vitamin C, ASA) is used as vitamin and antioxidant in the pharmaceutical and food industries. The synthesis of ASA has been of considerable interest for many years in view of the relatively large market capacity and high value as a special chemical.

In 1934, the Reichstein-Grussner method was first disclosed, a chemical synthetic route from glucose to ASA (Helv. Chim. acta 17: 311-328). Lazarus et al (1989, "Vitamin C: Bioconversion via Recombinant DNAapparatus", Genetics and Molecular Biology of Industrial microorganisms, American Society for Microbiology, Washington D.C., eds. C.L. Hershberger) disclose a Bioconversion process to produce the ASA intermediate 2-keto-L-gulonic acid (2-KLG, KLG) which can be chemically converted to ASA. The biological conversion of carbon sources to KLG involves a variety of intermediates, an enzymatic process associated with cofactor-dependent 2, 5-DKG reductase activity (2, 5-DKGR or DKGR).

It has been found that many bacterial species comprise DKGR, in particular members of the Corynebacterium (Corynebacterium) class, including Corynebacterium (Corynebacterium), Brevibacterium (Brevibacterium) and Arthrobacter (arthromobacter). Gridley et al (1988) Applied and Environmental Microbiology 54: 1770-1775 describes DKGR obtained from the Corynebacterium strain SHS 752001. U.S. Pat. No. 5,008,193 to Anderson et al discloses DKGR of Erwinia herbicola (Erwinia herbicoloa). Us patent 5,795,761; 5,376,544, respectively; 5,583,025, respectively; 4,757,012, respectively; 4,758,514, respectively; 5,004,690 and 5,032,514 disclose other reductases.

Host cells have been described in which enzymes involved in glycolysis have mutations. Harrod et al (1997) J.Ind.Microbiol.Biotechnol.18: 379-383; wedlock et al (1989) j.gen.microbiol.135: 2013 and 2018; and Walsh et al (1983) j.bacteriol.154: 1002-1004 describes a yeast having a glucokinase mutation. Glucokinase deficient bacteria have been described. Japanese patent publication JP4267860 describes a glucokinase-deficient Pediococcus sp. Russell et al (1989) appl.environ.microbiol.55: 294-297 describes Bacillus sphaericus (Bacillus sphaericus) deficient in glucokinase. Barredo et al (1988) Antimicrob. Agents-Chemother 32: 1061-1067 describes a glucokinase deficient strain of Penicillium chrysogenum. DiMarco et al (1985) appl.environ.microbiol.49: 151-157 describes mutants of Zymomonas mobilis deficient in glucokinase. Bouvet et al (1989) International Journal of systematic bacteriology, pages 61-67, describe bacteria that ferment glucose via the enberden-Meyerhoff pathway (Embden-Meyerhof pathway), such as members of the Enterobacteriaceae (Enterobacteriacea) and Vibrionaceae (Vibrionaceae) families. Truesdell et al (1991) Journal of Bacteriology, 6651-6656 describe the ketoaldonic acid (ketoaldonic acid) metabolic pathway of Erwinia (Erwinia sp.).

However, most existing methods for generating compounds overexpress the product. The problem of switching of the substrate used to produce the desired end product by the cell for metabolic (catabolic) purposes still remains, reducing the efficiency and its overall yield. Thus, there remains a need for improved methods of producing products via pathways related to metabolic pathways of host cells. The present invention addresses this need.

All publications cited herein are incorporated by reference in their entirety.

Summary of The Invention

The present invention provides a process for producing an ascorbic acid intermediate comprising genetically engineering a host cell that decreases carbon substrate diversion to a metabolic pathway, thereby increasing host cell yield.

In one embodiment, the present invention provides a method for enhancing the production of an ascorbic acid intermediate from a carbon source comprising,

a) obtaining an altered bacterial strain comprising an inactivated chromosomal gene of abacterial host strain,

b) cultivating said altered bacterial host strain under suitable conditions, and

c) producing an ascorbic acid intermediate from a carbon source, wherein the production of the ascorbic acid intermediate is enhanced over the production of the ascorbic acid intermediate in an unaltered bacterial host strain.

The ascorbic acid intermediate may be selected from gluconic acid, 2-keto-D-gluconic acid, 2, 5-diketo-D-gluconic acid, 2-keto-L-gulonic acid, L-iduronic acid, erythorbic acid, and tartaric acid. In a particular embodiment, the intermediate is 2, 5-diketo-D-gluconic acid. The altered bacterial strain can be obtained by inactivating the glk chromosomal gene. The altered bacterial strain may also be obtained by inactivating the gntk chromosomal gene. The altered bacterial strain can also be obtained by inactivating the glk and gntk chromosomal genes. The bacterial strain may be Pantoea (Pantoea), more specifically Pantoea citrea (Pantoea citrea).

In one embodiment, the host cell encodes a polynucleotide having an activity that phosphorylates D-glucose at the 6 th carbon and/or a polynucleotide encoding an activity that phosphorylates D-glucose at the 6 th carbon. The host cell is cultured in the presence of a carbon source and the yield of the product is demonstrated to be increased by direct and/or indirect assays. In certain embodiments of the invention, the method comprises a host cell having an alteration in a polynucleotide encoding an enzymatic activity for phosphorylating D-glucose at the 6 th carbon, and/or an alteration in a polynucleotide encoding an enzymatic activity for phosphorylating D-gluconate at the 6 th carbon.

Accordingly, in one aspect the present invention provides a method for producing an ascorbic acid intermediate in a recombinant host cell, comprising culturing a host cell capable of producing said ascorbic acid intermediate in the presence of glucose or a carbon source convertible by the host cell to glucose under conditions suitable for the production of said ascorbic acid intermediate, wherein said host cell can be modified to reduce diversion of glucose to a catabolic pathway of the organism. The modification may comprise inactivation or deletion of at least one polynucleotide encoding an enzymatic activity that diverts carbon source substrate to the catabolic pathway such that the level of the enzymatic activity is affected accordingly during the culturing.

In certain embodiments of the invention, the host cell further comprises at least one polynucleotide encoding an enzymatic activity that phosphorylates D-gluconate at the 6 th carbon, wherein said polynucleotide has been modified such that during culture the level of said enzymatic activity is altered, inactivated, or the amount of substrate transported across the cell membrane and/or phosphorylated for utilization by the cellular catabolic pathway is reduced. In certain embodiments, the process comprises the step of recovering the product, and in other embodiments, the process comprises the step of converting the product to another product.

In certain embodiments, the level of endogenous enzymatic activity is modulated during culturing, for example, by transcriptional or translational regulatory elements. Thus, in certain embodiments, an encoding polynucleotide that phosphorylates D-glucose for enzymatic activity transported across a cell membrane is operably linked to a regulatable promoter. In other embodiments, the polynucleotide encoding the enzymatic activity of carbon phosphorylating D-gluconate at position 6 is operably linked to a regulatable promoter. In other embodiments, the endogenous enzymatic activity is inactivated, e.g., by mutating or deleting part or all of the enzymatic activity encoding polynucleotide.

The present invention encompasses the production of ascorbic acid intermediates including gluconic acid, 2-keto-D-gluconic acid (2-KDG or KDG); 2, 5-diketo-D-gluconic acid (2, 5DKG or DKG); 2-keto-L-gulonic acid (2KLG or KLG); or L-Iduronic Acid (IA). In certain embodiments, the ascorbic acid intermediate is KLG, and the KLG is converted to ascorbic acid. In other embodiments, the ascorbic acid intermediate is KDG and KDG is converted to erythorbic acid.

The present invention provides a recombinant host cell capable of producing an ASA intermediate, wherein the host cell comprises a polynucleotide encoding an enzymatic activity that phosphorylates D-glucose at the 6 th carbon, wherein the polynucleotide is modified. The present invention also provides a recombinant host cell further comprising a polynucleotide encoding the enzymatic activity of carbon phosphorylating D-gluconate at position 6, wherein said polynucleotide is modified.

In certain embodiments, the host cell is a gram-positive microorganism, while in other embodiments, the host cell is a gram-negative microorganism. In certain embodiments, the gram-negative host cell is an Enterobacteriaceae (Enterobacteriaceae) host cell, including Erwinia (Erwinia), Enterobacteriaceae (Enterobacteriaceae), Gluconobacter (Gluconobacter), Acetobacter (Acetobacter), corynebacterium (corynebacterium), Escherichia (Escherichia), Salmonella (Salmonella), Klebsiella (Klebsiella), or Pantoea (Pantoea).

In other embodiments, the host cell may be native or appropriately genetically modified to be capable of utilizing a carbon source to maintain certain cellular functions (e.g., without limitation, with NAD, FADH₂Or NADPH form to produce reducing power), and will otherwiseAny bacterium that converts a carbon source into one or more products of commercial interest.

In certain embodiments, the enzymatic activity that phosphorylates D-glucose at the 6 th carbon comprises glucokinase, hexokinase, phosphotransferase system (PTS), or any other enzyme not necessarily classified in the above three groups but capable of such action. For example, an enzyme classified as a fructokinase because its preferred substrate is fructose, has the potential to phosphorylate D-glucose at a measurable rate. It is not uncommon for enzymes to act on other similar substrates, particularly high levels of substrates.

In other embodiments, the enzymatic activity that phosphorylates D-gluconic acid at the 6 th carbon comprises gluconokinase, deoxygluconate kinase, hexokinase, the phosphotransferase system (PTS), or any other enzyme not necessarily classified in group 4 above but capable of such action. For example, an enzyme classified as deoxygluconate kinase because its preferred substrate is deoxygluconate, has the potential to phosphorylate D-gluconate at a measurable rate. It is not uncommon for enzymes to act on other similar substrates, particularly high levels of substrates.

The invention also provides methods for producing host cells having altered levels of enzymatic activity. The present invention also provides novel nucleic acid and amino acid sequences for the enzymatic activity of phosphorylating D-glucose at the 6 th carbon and the enzymatic activity of phosphorylating D-gluconate at the 6 th carbon.

The invention also provides methods for producing host cells capable of producing different biomolecules of industrial value derived from glucose and/or fructose.

The invention also provides host cells having altered levels of enzymatic activity. The present invention also provides novel nucleic acid and amino acid sequences for the enzymatic activity of phosphorylating D-glucose at the 6 th carbon and the enzymatic activity of phosphorylating D-gluconate at the 6 th carbon.

The invention also provides host cells capable of producing different biomolecules of industrial value derived from glucose and/or fructose.

Brief Description of Drawings

FIG. 1 provides a schematic representation of some metabolic pathways involved in glucose assimilation in Pantoea citrea. X represents an enzymatic step affected by the genetic modification according to the invention. Boxes labeled with T represent putative transporters.

FIG. 2. some of the possible catabolic pathways that may be used to direct glucose into the cell metabolism. Arrows represent at least one enzymatic step.

FIG. 3 depicts products that can be obtained from the indicated commercial routes. Most of the carbon source used for the synthesis of the compounds listed on the left can be obtained from the catabolic pathway or the TCA cycle. In contrast, the majority of the carbon source of the right-hand compound is from the pentose pathway and/or from the oxidation of glucose to keto acids.

FIG. 4 depicts the nucleic acid sequence of Pantoea citrea glucokinase (glk) (SEQ ID NO: 1).

FIG. 5 depicts the amino acid sequence (SEQ ID NO: 2) of Pantoea citrea glucokinase (Glk).

FIG. 6 depicts the nucleic acid sequence (SEQ ID NO: 3) of Pantoea citrea gluconate kinase (gntk).

FIG. 7 depicts the amino acid sequence (SEQ ID NO: 4) of Pantoea citrea gluconate kinase (Gntk).

FIG. 8 depicts the amino acids of the genes glk 30, glk 31, gnt 1, gnt 2, pcgnt 3 and pcgnt 4 (SEQ ID NOS: 8-13).

FIG. 9 depicts D-glucose, D-gluconic acid, and certain derivatives thereof. The standard numbering of the carbons on glucose is shown by the numbers 1 and 6. 2-KDG ═ 2-keto-D-gluconic acid; 2, 5-DKG ═ 2, 5-diketogluconic acid; 2KLG ═ 2-keto-L-gulonic acid.

FIG. 10 depicts a general strategy for disrupting the gluconokinase gene of Pantoea citrea.

Figure 11 depicts the oxidation pathway for the production of ascorbic acid. E1 represents glucose dehydrogenase; e2 represents gluconate dehydrogenase; e3 represents 2-keto-D-gluconate dehydrogenase; and E4 represents 2, 5-diketo-D-gluconate reductase.

FIG. 12 depicts the net reaction (net reaction) of a host cell fermentation process capable of producing an ascorbic acid intermediate.

FIG. 13 depicts Carbon Evolution Rate (CER) and Oxygen Uptake Rate (OUR) of fermentation following exposure of wild-type organisms to glucose.

FIG. 14 depicts CER and OUR for single deletion (glucokinase) fermentation.

FIG. 15 depicts CER and OUR for single deletion (gluconokinase) fermentation.

FIG. 16 depicts CER and OUR for fermentation of host cells deficient in glucokinase and gluconokinase.

FIG. 17 illustrates the interrelationship of various metabolic pathways (including the glycolysis pathway, the TCA cycle, and the pentose pathway) with the oxidation pathway. Glk ═ glucokinase; gntk ═ gluconokinase; IdnO ═ iduronate reductase; IdnD ═ iduronate dehydrogenase; TKT ═ transketolase; TAL ═ transaldolase, 2KR ═ 2-ketoreductase; 2, 5DKGR ═ 2, 5-diketogluconate reductase.

FIG. 18 illustrates the correlation of various central metabolic pathways with modifications that will increase gluconic acid production. X represents the increased enzymatic pathway to be modified to effect gluconation.

FIG. 19 illustrates the correlation of various central metabolic pathways with modifications that will increase erythorbic acid production. X represents an increased enzymatic pathway to be modified to achieve the required production of erythorbic acid.

FIG. 20 illustrates the correlation of various central metabolic pathways with modifications that will increase ribose production. X represents the increased enzymatic or transport pathway to be modified to achieve the desired production of 2, 5-diketogluconate.

FIG. 21 illustrates the correlation of various central metabolic pathways with modifications that will increase tartaric acid production. X represents the required increased enzymatic step to be modified to achieve ribose production. IdnO ═ 5-keto-D-gluconic acid 5-reductase; IdnD ═ 1-iduronate 5-dehydrogenase.

FIG. 22 illustrates the pathway for dihydroxyacetone phosphate (DHAP) conversion to glycerol.

FIG. 23 depicts the primer DNA sequence for PCR amplification of a 2.9kb DNA fragment containing the glpK gene as described in example 7.

FIG. 24 depicts the DNA sequence of the Pantoea citrea glycerol kinase structural gene as described in example 7. The sequence used to disrupt the HpaI site of the gene is underlined.

FIG. 25 depicts the protein sequence of Pantoea citrea glycerol kinase as described in example 7.

Detailed Description

The present invention provides methods and host cells for producing ascorbic acid intermediates. Recombinant host cells have been constructed that produce ascorbic acid intermediates, as well as recombinant host cells genetically engineered to reduce carbon source substrate diversion tocatabolic pathways. In one embodiment, the recombinant host cell produces a modified level of enzymatic activity that is transported into the cell for a catabolic pathway, such as an enzymatic activity that phosphorylates D-glucose at the 6 th carbon and/or an enzymatic activity that phosphorylates D-gluconate at the 6 th carbon. The intracellular metabolism of glucose and/or gluconic acid is reduced when the recombinant cell is cultured in the presence of glucose or a carbon source capable of being converted to glucose by the host cell.

In certain embodiments, the host cell is a Pantoea citrea cell that has been genetically engineered to produce a product, such as an ascorbic acid ("ASA") intermediate. In these embodiments, the present invention provides the particular advantage of being able to utilize fermentation processes to produce ASA intermediates.

The present invention provides a further advantage in that extracellular oxidation of substrates is separated from metabolic pathways using these oxidation products.

The present invention provides another advantage in that the increase in production of the desired product is measured directly or the oxygen consumption or CO is measured indirectly₂Exhaust gas (CO)₂Production), demonstrating increased efficiency of ascorbic acid intermediate production compared to wild-type biotransformation.

Another advantage provided by the present invention is that the host cell is capable of producing ascorbic acid intermediates using two different carbon sources simultaneously.

General techniques

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry within the skill of the art. These techniques are well elucidated in the literature, for example, Molecular Cloning: ALaborory Manual, second edition (Sambrook et al, 1989); current protocols in Molecular Biology (F.M. Ausubel et al, 1987 and annual updates); Oligonucleotide Synthesis (M.J. Gait, 1984); and PCR: The Polymerase Chain Reaction, (Mullis et al, 1994); Manual of Industrial Microbiology and Biotechnology, second edition (A.L. Demain et al, 1999).

Definition of

As used herein, "host cell oxidative pathway" means that the host cell comprises at least one oxidase that oxidizes a carbon source such as D-glucose and/or a metabolite thereof. By "membrane" or "membrane-bound" glucose oxidation pathway in a host cell is meant that the host cell oxidizes a carbon source, such as D-glucose and/or a metabolite thereof, by at least one membrane-bound oxidase activity. In certain embodiments, the host cell oxidative pathway comprises an enzymatic activity. In other embodiments, the host cell oxidative pathway comprises two or more enzymatic activities.

As used herein, a "host cell catabolic pathway" means that the host cell comprises at least one enzymatic activity that produces ATP or NADPH, for example, by phosphorylating a carbon source (e.g., D-glucose and/or metabolites thereof). By a host cell "intracellular" catabolic pathway is meant that the host cell comprises at least one of the enzymatic activities in its cytoplasm. In certain embodiments, the host cell catabolic pathway comprises an enzymatic activity. In other embodiments, the host cell catabolic pathway comprises two or more enzymatic activities. Catabolic pathways include, but are not limited to, glycolysis, pentose pathway, and TCA pathway (see fig. 17).

The phrase "enzymatic transport system" as used herein refers to an enzymatic activity that consumes energy (ATP) and transports carbon substrates across cell membranes, typically by adding phosphate at the 6 th carbon of D-glucose, including the enzymatic activities of glucokinase (E.C. -2.7.1.2) and phosphotransferase system (PTS) (E.C. -2.7.1.69).

The phrase "enzymatic activity which phosphorylates D-gluconic acid at the 6 th carbon" as used herein refers to an enzymatic activity which phosphorylates D-gluconic acid at the 6 th carbon thereof, including the gluconokinase (E.C. -2.7.1.12) enzymatic activity.

The phrase "enzymatic activity phosphorylating D-glucose at the 6 th carbon" as used herein refers to an enzymatic activity of adding phosphate to the 6 th carbon of D-glucose, and includes glucokinase (E.C. -2.7.1.2) and phosphotransferase system (PTS) (E.C. -2.7.1.69) enzymatic activities.

The phrase "enzymatic activity which phosphorylates D-gluconic acid at the 6 th carbon" as used herein refers to an enzymatic activity which phosphorylates D-gluconic acid at the 6 th carbon thereof, including the gluconokinase (E.C. -2.7.1.12) enzymatic activity.

As used herein, a "modified" level of enzyme activity produced by a host cell or a "modified level" of enzyme activity of a host cell refers to controlling the level of enzyme activity produced during culture so that its level increases or decreases as desired. The desired change in the level of enzyme activity may be genetically engineered to produce one or both enzyme activities simultaneously or sequentially in any order. To control the level of enzyme activity, the host cell is genetically engineered such that the nucleic acid encoding the enzyme activity is under transcriptional or translational control.

The term "modified" as used herein in reference to a nucleic acid or polynucleotide means that the nucleic acid has been altered in some way, e.g., by mutation, from the wild-type nucleic acid; deletion of part or all of the nucleic acid; or by operably linking to a transcriptional control region. The term "mutation" as used herein in reference to a nucleic acid refers to any alteration in the nucleic acid such that the product of the nucleic acid is partially or completely inactivated or deleted. Examples of mutations include, but are not limited to, point mutations, frameshift mutations, and partial or complete deletions of the gene encoding the enzyme activity, e.g., an enzyme activity that transports a substrate across the cell membrane, such as an enzyme activity that phosphorylates D-glucose at its 6 th carbon or phosphorylates D-gluconate at its 6 th carbon.

An "altered strain" according to the present invention is a strain that produces an increased level of production of a genetically engineered bacterial microorganism under substantially the same growth conditions relative to the level of production of the same end product in a corresponding unaltered bacterial host strain. An "unaltered bacterial strain" or host is a bacterial microorganism in which the coding sequences that are diverted to the enzymatic pathway are not inactivated and retain enzymatic activity. The increased production level is caused by inactivation of one or more chromosomal genes. In one embodiment, the increased expression level is caused by a deletion of one or more chromosomal genes. In another embodiment, the increased expression level is caused by insertional inactivation of one or more chromosomal genes. Preferably, the inactivated gene is selected from the genes encoding enzymes to be inactivated as described herein. For example, in one embodiment, the one or more chromosomal genes are selected from glk and gntk.

In certain embodiments, the altered bacillus strain may comprise two inactivated genes, three inactivated genes, four inactivated genes, five inactivated genes, six inactivated genes, or more. The inactivated genes may be adjacent to each other or located in separate regions ofthe bacillus chromosome. An inactivated chromosomal gene may have a function that is essential under certain conditions, but the gene is not essential for the survival of the bacillus strain under laboratory conditions. Preferred laboratory conditions include, but are not limited to, conditions such as growth in a fermentor, shaken plates or plate media, and the like.

As used herein, the term "inactivation", when referring to an enzymatic activity, refers to removal of the activity by any means, including mutation, or partial or complete deletion of the nucleic acid encoding the enzymatic activity. The term "inactivation" includes any method of preventing the functional expression of one or more chromosomal genes of interest, wherein the gene or gene product is unable to perform its known function. The chromosomal gene of interest depends on the enzymatic activity that is intended to be inactivated. For example, inactivation of glucokinase and/or gluconokinase activity may be achieved by inactivation of the glk and/or gntk chromosomal genes. Inactivation may include such methods as deletion, mutation, disruption or insertion of the nucleic acid gene sequence. In one embodiment, the expression product of an inactivated gene may be a truncated protein, so long as the biological activity of the protein is altered. The alteration in biological activity may be an altered activity, but preferably a loss of biological activity. In the altered bacterial strains of the present invention, the inactivation of one or more genes is preferably a stable, irreversible inactivation.

In a preferred embodiment, the deletion of the gene is preferably by homologous recombination. For example, as shown in FIG. 9, if the glk gene is to be deleted, the chloramphenicol resistance gene is cloned into a restriction site in the glucokinase gene. Will Cm^RThe gene is inserted into the Pst I site of the coding region of the gene structure. Themodification was then transferred into Pantoea citrea glkA by homologous recombination using a non-replicating R6K vector^-In the chromosome(s) of (c). Cm is then removed from the glk coding region^RGenes, leaving a disrupted spacer region in the coding region, inactivate the coding region. In another embodiment, Cm is determined by replacing part of the coding region^RThe gene is inserted into the coding region. Cm is then removed^RA gene, without the reinsertion of a replacement portion of the coding region, results in the effective deletion of a portion of the coding region, inactivating that region.

A gene deletion as used herein may include deletion of the entire coding sequence, deletion of portions of the coding sequence, or deletion of the coding sequence including flanking regions. The deletion may be a partial deletion as long as the sequence remaining in the chromosome is too short for the biological activity of the gene. The flanking regions of the coding sequence may comprise about 1-500bp of the 5 'and 3' ends. The flanking region may be greater than 500bp, but preferably no other genes are included in the region which may be inactivated or deleted according to the invention. The net result is that the deleted gene is virtually nonfunctional.

In another preferred embodiment, inactivation is by insertion. For example, if glk is the gene to be inactivated, the DNA construct will contain an introduced sequence with the glk gene interrupted by a selectable marker. The selectable marker is flanked by fragments of the glk coding sequence. The DNA construct inactivates the glk gene by inserting a selectable marker in a double crossover event along with the substantially identical sequence arrangement of the glk gene in the host chromosome.

In another embodiment, the plasmid is used as a vector and inactivated by insertion in a single crossoverevent. For example, the glk chromosomal gene is arranged with a plasmid containing the gene or part of the gene coding sequence and a selectable marker. The selectable marker may be located within the coding sequence of the gene or in a part of a plasmid separate from the gene. The vector is integrated into the bacillus chromosome and the gene is inactivated by insertion of the coding sequence into the vector.

It may also be inactivated by genetic mutation. Methods for gene mutation are well known in the art and include, but are not limited to, chemical mutagenesis, site-directed mutagenesis, generation of random mutations, and gap-duplication (gapped-duplex) methods. (USP 4,760,025; Moring et al, Biotech.2: 646 (1984); and Kramer et al, Nucleic Acids Res.12: 9441 (1984)).

Inactivation can also occur by applying the above-described inactivation methods to the corresponding promoter regions of the genomic region of interest.

"under transcriptional control" or "transcriptional control" is a term well understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, is dependent on its operative linkage with an element that initiates, or initiates, transcription. "operably linked" refers to adjoining (juxtaposition) wherein the elements are in an arrangement such that they function.

The term "regulatable promoter" as used herein refers to a promoter element which can regulate its activity or function. Can be regulated in a variety of ways, most commonly by protein interactions, interfering with or increasing the ability of RNA polymerase to initiate transcription.

"under translational control" is a term well understood in the art to denote a regulatory process that occurs after the formation of mRNA.

The term "batch" as used herein describes a batch of cell cultures to which the substrate is initially added as a solid or concentrate at the start of a run. Batch culture starts with seeding the culture medium with cells, but in contrast to fed-batch (fed-batch) culture, no nutrients are fed subsequently, e.g. by a concentrated nutrient feed. In contrast to continuous culture, batch cell culture does not systematically add or remove culture fluid or cells from the culture. Since the concentration of nutrients and metabolites in the medium depends on the starting concentration of the batch, the composition of the added nutrients is subsequently altered by fermentation and thus the various analytes cannot be added subsequently to the medium.

The term "fed-batch" as used herein describes a batch of cell cultures to which a substrate is added periodically or continuously during the run, either as a solid or as a concentrated solution. Fed-batch culture also starts with seeding of the medium with cells as with batch culture, but with the difference that the nutrients are subsequently fed in, for example by concentrating the nutrient feed. Unlike continuous culture, fed-batch culture does not systematically remove the culture broth or cells, and is advantageous for related applications where the levels of various analytes in the medium are monitored and controlled, since the concentrations of nutrients and metabolites in the medium are easily controlled or easily influenced by feeding with varying nutrient components. The nutrient feed to the fed-batch culture is typically a concentrated nutrient solution comprising an energy source such as carbohydrates; the concentrated nutrient solution delivered to the fed-batch culture may optionally comprise amino acids, lipid precursors and/or salts. In fed-batch culture, the nutrient feed is typically concentrated to minimize the increase in culture volume when sufficient nutrients are supplied for continuous cell growth.

The term "continuous cell culture" orsimply "continuous culture" is used herein to describe a culture characterized by a continuous inflow of liquid nutrient feed and a continuous outflow of liquid. The nutrient feed may be, but is not necessarily, a concentrated nutrient feed. Nutrient solution was continuously replenished at approximately the same rate and the culture was maintained in a stable state of proliferation and growth by flushing the cells out of the reactor with spent medium. In a type of bioreactor known as a chemostat, the cell culture is continuously fed with fresh nutrient medium and the spent medium, cells and secreted cell products are continuously withdrawn. In addition, continuous culture may form a "perfusion culture" in which the effluent comprises a medium that is substantially free of cells, or substantially lower than the concentration of cells in the bioreactor. In perfusion culture, the cells may be retained by, for example, filtration, centrifugation, or sedimentation.

As used herein, "culturing" refers to the bioconversion of a carbon source substrate to the desired end product by fermentation in a reaction vessel. As used herein, bioconversion refers to the conversion of a carbon source substrate to an end product of interest by contacting the carbon source substrate with a microorganism.

As used herein, "oxygen uptake" or "OUR" refers to the determination of the specific oxygen consumption (specific consumption) within a reaction vessel. Various on-line tests can be used to determine oxygen consumption. In one embodiment, OUR (mmol/(liter-hour)) is determined as follows: (air flow (liter/min)/fermentation weight (fermentation broth in kg) X for O₂X broth density X (calibration constant for 21.1C and standard 20.0C air flow calibration)) - ([ air flow/fermentation weight)]X [ exhaust gas O₂Waste gas N₂]X for N₂X broth density X constant).

As used herein, "carbon release rate" or "CER" refers to the determination of production in a reaction vessel during fermentationHow much CO₂. Usually because CO is not supplied to the reaction vessel initially or subsequently₂Any CO₂Are assumed to result from fermentation processes occurring within the reaction vessel. "exhaust gas CO₂"refers to CO typically determined in a reaction vessel using mass spectrometry methods known in the art₂The number of the cells.

As used herein, "yield" refers to product/substrate amount. Yields can be expressed as weight% (product gm/substrate gm) or as moles of product/moles of substrate. For example, the amount of substrate, e.g., glucose, can be determined by the feed rate and the concentration of added glucose. The amount of product present is determined by various spectrophotometric or analytical methods. One such method is High Performance Liquid Chromatography (HPLC). Increased yield refers to an increased yield compared to the conversion yield using the wild-type organism, e.g. 10%, 20% or 30% increase over the wild-type yield.

The phrase "production enzyme" as used herein refers to an enzyme or enzyme system that can catalyze the conversion of a substrate to a product of interest. Production enzymes include, but are not limited to, oxidases and reductases.

The phrase "oxidase enzyme" as used herein refers to an enzyme or enzyme system that can catalyze the conversion of a substrate in a particular oxidation state to a product in a higher oxidation state than the substrate. As used herein, the phrase "reductase" refers to an enzyme or enzyme system that can catalyze the conversion of a substrate in a particular oxidation state to a product in a lower oxidation state than the substrate. In an illustrative example disclosed herein, the oxidases involved in biocatalytic D-glucose or metabolites thereof in pantoea cells that have been engineered to produce ASA intermediates include D-glucose dehydrogenase, D-gluconate dehydrogenase and 2-keto-D-gluconate dehydrogenase. In another illustrative embodiment disclosed herein, the reductases associated with biocatalytic D-glucose or metabolites thereof in pantoea cells that have been engineered to produce ASA intermediates as described herein include 2, 5-diketo-D-gluconate reductase, 2-ketoreductase and 5-ketoreductase. Such enzymes include those naturally produced by the host strain or introduced by recombinant means.

The term "carbon source" as used herein encompasses suitable carbon sources commonly used by microorganisms, such as 6-carbon sugars, including but not limited to the D or L forms of glucose, gulose, sorbose, fructose, idose, galactose and mannose, or combinations of 6-carbon sugars, such as glucose and fructose, and/or 6-carbon sugar acids, including but not limited to 2-keto-L-gulonic acid, idonic acid, gluconic acid, 6-phosphogluconic acid, 2-keto-D-gluconic acid, 5-keto-D-gluconic acid, 2-keto-glucuronic acid phosphate, 2, 5-diketo-L-gulonic acid, 2, 3-L-diketo-gulonic acid, dehydroascorbic acid, erythorbic acid and D-mannonic acid, or enzymatic derivatives thereof.

The following abbreviations are used herein for D-glucose or glucose (G); d-gluconic acid or Gluconic Acid (GA); 2-keto-D-gluconic acid (2 KDG); 2, 5-diketo-D-gluconic acid (2, 5DKG or DKG); 2-keto-L-gulonic acid (2KLG or KLG); L-Iduronic Acid (IA); erythorbic Acid (EA); ascorbic acid (ASA); glucose Dehydrogenase (GDH); gluconate dehydrogenase (GADH); 2, 5-diketo-D-gluconate reductase (DKGR); 2-keto-D-gluconate reductase (KDGDH); d-ribose (R); 2-ketoreductases (2KR or KR) and 5-ketoreductases (5KR or KR).

By "allowing the production of an ascorbic acid intermediate from a carbon source, wherein the production of the ascorbic acid intermediate is enhanced compared to the production of the ascorbic acid intermediate in an unaltered bacterial host strain" is meant that the altered bacterial strain contacts a substrate, e.g. a carbon source, to produce the end product of interest. The inventors have found that by inactivating genomic expression and thereby altering certain enzymatic activities, the microorganism shows enhanced production of the end product.

As used herein, "desired end product" refers to the compound of interest into which the carbon source substrate is biologically converted. The desired end product may be the actual compound desired or an intermediate of another pathway. Typical end products of interest are listed on the right side of figure 3.

The term bacteria as used herein refers to any group of prokaryotic, i.e. micro-organisms lacking membrane-bound nuclei and organelles. All bacteria are encapsulated by lipid membranes that regulate the entry and exit of substances into and out of the cells. The rigid cell wall completely surrounds the bacteria and is located outside the membrane. There are many different types of bacteria, some of which include, without limitation, strains of the enterobacteriaceae, bacillus, streptomyces, pseudomonas, and erwinia families.

As used herein, the "Enterobacteriaceae" family refers to bacterial strains having the general characteristics of gram-negativity and being facultative anaerobic, including the genera Escherichia (Escherichia coli), Shigella (Shigella), Edwardsiella (Edwardsiella); salmonella (Salmonella), Citrobacter (Citrobacter), Klebsiella (Klebsiella), Enterobacter (Enterobacter), Serratia (Serratia), Proteus (Proteus), Morganella (Morgarella), Providencia (Providecia), and Yersinia (yersinia). For the production of ASA intermediates, preferred strains of the Enterobacteriaceae family are those which are capable of producing 2, 5-diketo-D-gluconic acid from D-glucose or from a carbon source which can be convertedby the strain into D-glucose. Enterobacteriaceae families capable of producing 2, 5-diketo-D-gluconic acid from D-glucose solutions include, for example, Erwinia, Enterobacter, Gluconobacter, and Pantoea. Intermediates of the microbial pathway from a carbon source to ASA include, but are not limited to, GA, KDG, DKG, KLG, IA and EA. The preferred Enterobacteriaceae fermenting strains of the present invention for producing ASA intermediates are Pantoea species, in particular Pantoea citrea. Other strains of the enterobacteriaceae family that produce ASA intermediates include, but are not limited to, escherichia coli and gluconobacter spp.

The "Bacillus" family as used herein refers to strains of bacillary bacteria having the general characteristics of gram-positivity, capable of producing spores under certain environmental conditions.

The term "recombinant" as used herein refers to a host cell that has genomic alterations, e.g., by addition of a nucleic acid that does not naturally occur in the organism, or by alteration of a nucleic acid that naturally occurs in the host cell, including host cells that have additional copies of an endogenous nucleic acid introduced by recombinant means. The term "heterologous" as used herein refers to a nucleic acid or amino acid sequence that does not occur naturally in a host cell. The term "endogenous" as used herein refers to a nucleic acid that occurs naturally in a host.

The term "isolated" or "purified" as used herein refers to an enzyme, or a nucleic acid, or a protein, or a peptide, or a cofactor, which is separated from at least one component with which it is naturally associated. In the present invention, an isolated nucleic acid may include a vector comprising the nucleic acid.

It is well understood in the art that acidic derivatives of sugars have various ionization states, depending on the environmental medium, outside the solutions they are prepared in, if in solution, or if in solid form. The use of terms such as iduronic acid to refer to the above molecules is intended to include all ionization states of the organic molecule. Thus, for example, "iduronic acid," its cyclized form "idonolactone" and "iduronate ester" refer to the same organic moiety and are not intended to indicate a particular ionization state or chemical form.

The term "vector" as used herein refers to a polynucleotide construct designed to transduce/transfect one or more cell types, including, for example, a "cloning vector" designed to isolate, propagate, and replicate an inserted nucleotide, or an "expression vector" designed to express a nucleotide sequence in a host cell, such as a Pantoea citrea or E.coli host cell.

The terms "polynucleotide" and "nucleic acid" are used interchangeably herein to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. These terms include single-, double-, or triple-stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases, or other natural, chemical, biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the polynucleotide may comprise sugars and phosphate groups (as typically found in RNA or DNA), or modified or substituted sugars or phosphate groups. In addition, the backbone of the polynucleotide may comprise synthetic subunits such as polymers of phosphoramidates and thus may be phosphoramidate (P-NH2) or mixed phosphoramidate-phosphodiester oligomers of oligodeoxynucleosides. Peyrottes et al (1996) nucleic acids Res.24: 1841-8; chaturvedi et al (1996) Nucleic acids sRes.24: 2318-23; schultz et al (1996) Nucleic Acids Res.24: 2966-73. Phosphorothioate linkages may be used in place of phosphodiester linkages. Braun et al (1988) J.Immunol.141: 2084-9; latimer et al (1995) Molec.Immunol.32: 1057-1064. In addition, double-stranded polynucleotides can be obtained from chemically synthesized single-stranded polynucleotide products by synthesizing complementary strands and annealing under appropriate conditions, or by de novo synthesis of complementary strands using a DNA polymerase and appropriate primers. The relevant polynucleotide sequences (e.g.the relevant SEQ ID NO) also include complementary sequences.

The following are non-limiting examples of polynucleotides: genes or gene fragments, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may comprise modified nucleotides (e.g., methylated nucleotides and nucleotide analogs), uracyl, other sugars and linking groups (e.g., fluorinated ribose and thioate), and nucleotide branches. The nucleotide sequence may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, for example by conjugation of a labeling element. Other types of modifications included within this definition are capping, replacing one or more naturally occurring nucleotides with an analog, and introducing means for attaching the polynucleotide to a protein, metal ion, labeling element, other polynucleotide, or solid support. Preferably, the polynucleotide is DNA. As used herein, "DNA" includes not only bases A, T, C and G, but also any analogs or modified forms of these bases, such as methylated nucleotides, nucleoside internal modifications (e.g., uncharged linkages and thioates), the use of sugar analogs, and modifications and/or alterations to the backbone structure (e.g., polyamides).

A polynucleotide or region of a polynucleotide has a certain percentage "sequence identity" (e.g., 80%, 85%, 90%, 95%, 97%, or 99%) to another sequence, meaning that when aligned, the percentage of identical bases in the two sequences are compared. This alignment, as well as percent homology or sequence identity, can be determined using software programs known in the art, such as those described in Current protocols in Molecular Biology (F.M. Ausubel et al, 1987) suppl.30, 7.7.18. The preferred alignment program is ALIGN Plus (Scientific and economic Software, Pennsylvania), preferably using the following default parameters: mismatch is 2; open gap is 0; the extension gap is 2.

The polynucleotide sequence "described" in SEQ ID NO refers to the same contiguous sequence as shown in SEQ ID NO. The term encompasses a portion or region of the SEQ ID NO as well as the entire sequence contained within the SEQ ID NO.

"expression" includes transcription and/or translation.

As used herein, the term "comprising" and its cognates are used in their inclusive sense; that is, to the extent that the term "includes" or its cognate terms are used, such terms are intended to be inclusive.

The terms "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

Enzyme activity

Trisaccharides, tetrasaccharides, pentoses, hexoses and heptoses form phosphate esters. The initial step in all sugar metabolism is its phosphorylation. Whereby glucose is phosphorylated to glucose-6-phosphate. All cells that can metabolize glucose contain some form of hexokinase that catalyzes the reaction

FIG. 9 depicts D-glucose and shows the "carbon 6". Typical hexokinases include hexokinase (Frohlich et al, 1985, Gene 36: 105-. The DNA sequence of the structural gene of citrullinated glucokinase is shown in FIG. 4. Recognition sites for the restriction enzymes NcoI (CCATGG) and SnaBI (TACGTA) are marked. FIG. 5 depicts the protein sequence of the Pantoea citrea glucokinase gene. Most hexokinases are somewhat non-specific and have been shown to have some ability to catalyze the formation of mannose, fructose and galactose 6-phosphates. In addition, other hexose derivatives may also be phosphorylated by hexokinase. For example, gluconic acid (FIG. 3) may also be phosphorylated by kinases, particularly gluconic acid kinase (cited). The sequence of the gluconokinase structural gene of Pantoea citrea is shown in FIG. 6. The recognition site for the restriction enzyme Pst I (CTGCAG) is highlighted. The protein sequence of the gluconokinase gene of Pantoea citrea is shown in FIG. 7 (SEQ ID NO 4). The genes for certain glucokinases and gluconokinases (glk, gntk, etc.) are shown in FIG. 8.

FIG. 17 shows the correlation between catabolic and oxidative pathways. Glucose can be phosphorylated to glucose-6-phosphate by glucokinase (Glk), entering the catabolic pathway through the glycolytic pathway; and phosphorylation of gluconic acid to gluconic acid-6-phosphate by gluconokinase (Gntk) through the pentose pathway into the catabolic pathway. Inactivation or modification of the levels of glucokinase and gluconokinase by modification of their (glk and/or gntk) encoding nucleic acids or polypeptides results in increased yields of the desired product, e.g., an ascorbic acid intermediate. Genetic modifications are used to eliminate the link between catabolic functions and the enzymatic reactions required for synthesis of the desired product. Although in one embodiment glucokinase and gluconokinase are modified, the inventors are concerned with modifying other enzymatic steps to separate them from the oxidative, catabolic pathways.

In another embodiment, the catabolic pathway is separated from the production pathway to increase the production of 5-KDG and/or tartaric acid. As shown in FIG. 21, glucose can enter the catabolic pathway through the glycolytic pathway, e.g., through glucose-6-phosphate, through the glucose-6-phosphate and other ascorbic acid by-products, e.g., the pentose pathway of iduronic acid and 2-KLG. Inactivation or modification of the levels of glucokinase, gluconokinase, 2, 5-DKG reductase, 5-keto-D-gluconate 5-reductase (idnO) and idose 5-dehydrogenase (idnD) by modifying the nucleic acids or polypeptides encoding them results in an increased yield of the product of interest, e.g., 5-DKG and/or tartaric acid.

In another embodiment, the catabolic pathway is separated from the production pathway to increase the production of gluconic acid. As shown in FIG. 18, glucose can enter the catabolic pathway through the glycolytic pathway, e.g., through the glucose-6-phosphate, and through the pentose pathway of gluconate-6-phosphate. Inactivation or modification of the levels of glucokinase, gluconokinase and glyceraldehyde hydrogenase by modification of their encoding nucleic acids or polypeptides results in an increased yield of the desired product, e.g. gluconic acid.

In another embodiment, the catabolic pathway is separated from the production pathway to increase the production of erythorbic acid. As shown in FIG. 19, glucose can be produced by the glycolytic pathway, e.g., by glucose-6-phosphate; the pentose pathway through glucono-6-phosphate; and transporting the 2-KDG and the 2, 5-KDG to the cytoplasm via an enzymatic transport system, thereby accessing a catabolic pathway. Inactivating or modifying the levels of glucokinase, gluconokinase and 2-KDG hydrogenase by modifying the nucleic acid or polypeptide encoding them; and 2-KDG into the cytoplasmic transport system, resulting in increased yields of the desired product, e.g., erythorbic acid.

In another embodiment, the catabolic pathway is separated from the production pathway to increase the production of 2, 5-DKG.As shown in FIG. 20, glucose can be produced by the glycolytic pathway, e.g., by glucose-6-phosphate; the pentose pathway through glucono-6-phosphate; and transporting the 2-KDG and the 2, 5-KDG to the cytoplasm via an enzymatic transport system, thereby accessing a catabolic pathway. Inactivating or modifying the levels of glucokinase, gluconokinase and 2-KDG hydrogenase by modifying the nucleic acid or polypeptide encoding them; and 2-KDG into the cytoplasmic transport system, resulting in an increased yield of the desired product, e.g., 2, 5-DKG.

Recombinant techniques are available that affect the expression of enzymes in a foreign host, allowing this aspect of the invention to be accomplished, which envisions the production of ascorbic acid intermediates from readily available carbon source substrates by diverting the carbon substrate reduction to catabolic pathways. This method is considerably superior to existing methods and is characterized by a reduced number of substrates that switch to catabolic pathways and are thus not converted to the desired oxidative end product, such as ascorbic acid intermediates. This results in increased fermentation efficiency and yield compared to fermentation with wild-type organisms. Certain wild-type organisms may produce ascorbic acid intermediates, such as 2-KLG, however the level of production may not be sufficient to be economically feasible. It has been noted that wild-type pantoea citrifolia has its own cytoplasmic glucokinase and gluconokinase, which enables the organism to convert glucose into phosphorylated derivatives for use in the central metabolic pathway, and which generates the necessary energy-consuming ATP, resulting in more carbon being diverted to the non-2-KLG production pathway. Using the method of the present invention under the same control conditions in two disrupted plasmids described further herein, as described below, the glucokinase and gluconokinase genes can be deleted from the genome of Pantoea citrea, such that the modified Pantoea citrea is capable of producing an increased level of DKG from glucose as compared to the wild-type level, e.g., from 63% to about 97-98% yield level. [ see example 6].

The method of generating the single organism transformation encompassed by the present invention comprises constructing an expression vector for the missing kinase described above, and transferring the vector into cells capable of initially converting the general metabolite into the 2, 5-DKG substrate of the enzyme by any of the gene transfer methods described above, such as transformation, transduction, or conjugation. As described in the examples below, gene transfer results in organisms with increased yield of ascorbic acid intermediates. The specification describes details of vector construction, gene transfer, and use of the resulting organisms.

Alternatively, a gene encoding an enzyme known to affect the conversion of glucose or other general metabolites to 2, 5-DKG can be cloned from an organism known to contain the gene, an expression vector containing the cloned gene sequence can be constructed, and the vector can be transferred to cells that normally produce 2, 5-DKG reductase. Examples of enzymes which influence the conversion of a typical metabolite into 2, 5-DKG are D-glucose dehydrogenase (Adachi, O. et al, Agric.biol. chem., 44 (2): 301-308[1980]Ameyama, M. et al, Agric.biol. chem.45[4]: 851 861[1981]), D-gluconate dehydrogenase (McIntire, W. et al, biochem.J., 231: 651-654[1985]; Shinagawa, E. et al, Agric.biol. chem.40[3]: 475-483[1976]; Shinagawa, E. et al, Agric.biol. chem.42[5]: 1055-1057[1978]), 5-keto-D-gluconate dehydrogenase and 2-D-gluconate dehydrogenase (Biond 1085. chem.1085, Biond.1085. chem.1085, Biol.. The third method is to transfer the complete sequence comprising the enzyme transforming the general metabolite 2-KLG into a neutral host. The advantage of this last method is that the host organism can be chosen almost arbitrarily, irrespective of its possible any desired growth characteristics and nutritional requirements. Thus, the use of organisms with appropriate culture and growth experience, such as E.coli and Bacillus, as host cells has the advantage of consistency over other methods in terms of bacterial production of enzymes or substrates.

Once an organism capable of transformation has been produced, the methods of the invention can be carried out in a variety of ways depending on the nature of the recombinase expression vector construction and the growth characteristics of the host. Host organisms are typically grown under conditions conducive to the production of large numbers of cells. When a large number of cells accumulate, the promoter provided by the recombinant gene sequence becomes or is already active, and can transcribe and translate the coding sequence. When these genes are properly expressed and thus the desired catalytic amount of enzyme is present, starting material such as glucose is added to the medium at a level of 1-500g/L and the culture is maintained at about 20-40℃, preferably about 25-37℃, for 1-300 hours until conversion to 2-KLG is achieved. The concentration of starting material may be maintained at a constant level by continuous feed control, and the 2-KLG produced may be recovered from the medium in batches or continuously using methods known in the art.

C. General methods to which the invention relates

In the following examples, the following general methods are used in connection with probe construction, screening, hybridization of probes to targets, and vector construction.

C.1 plasmid isolation, restriction enzyme cleavage

Clewell, d.b. and Helinski, Biochemistry 9: 4428(1970), plasmid isolation from the identified cultures and purification by Biorad A-50 Agarose column chromatography. Birnboim, h.c. nucleic acids Research 7: 1513(1979) mini-preps.

Treating about 20.mu.g of plasmid with 10-50 units of a suitable restriction enzyme or series of restriction enzymes in about 600.mu.l of a solution of an appropriate buffer or series of buffers containing the restriction enzyme used; each enzyme was incubated at 37 ℃ for 1 hour, and fragments of the cloned plasmids were prepared for sequencing. After incubation with each enzyme, the proteins were removed by phenol-chloroform extraction and ethanol precipitation to recover the nucleic acids.

As shown in fig. 1, there are multiple links to the catabolism, pentose and tricarboxylic acid (TCA) pathways. GDH ═ glucose dehydrogenase; GADH-gluconate dehydrogenase; 2-kDGH ═ 2-keto-d-gluconate dehydrogenase; 2-KDG ═ 2-keto-D-gluconic acid; IADH ═ iduronate dehydrogenase; 2, 5-DKG ═ 2, 5-diketogluconic acid; 2KLG ═ 2-keto-L-gulonic acid; 5-KDG ═ 5-keto-D-gluconic acid; 2KR ═ 2-ketoreductase; 2, 5DKGR ═ 2, 5-diketogluconate reductase; GIkA ═ glucokinase; GntK ═ gluconokinase; 5-KR ═ 5-ketoreductase; 2, 5-DKGR. It can be seen that removal of glucokinase activity blocks glucose entry into the catabolic pathway, whereas deletion of the gluconokinase activity does not allow gluconic acid to enter the pentose assimilation pathway. It is evident from this figure that the lack of glucokinase and/or gluconokinase activity impairs the metabolism of glucose and its oxidation products.

Production of ASA intermediates

The present invention provides methods for producing ascorbic acid intermediates in host cells. The invention comprises a method of reducing the level of enzymatic activity that phosphorylates D-glucose at the 6 th carbon and/or the level of enzymatic activity that phosphorylates D-gluconate at the 6 th carbon during part orall of the culturing period. The invention comprises a method for increasing the level of enzymatic activity that phosphorylates D-glucose at the 6 th carbon and/or the level of enzymatic activity that phosphorylates D-gluconate at the 6 th carbon during part or all of the cultivation. The invention also encompasses a method wherein the level of enzymatic activity that phosphorylates D-glucose at the 6 th carbon and/or the level of enzymatic activity that phosphorylates D-gluconate at the 6 th carbon is not altered or increased at the start of culture to promote growth, i.e.the production of cellular biomass, and decreased later in culture to promote accumulation of the product of interest.

The present invention provides altered nucleic acids or polynucleotides that separate oxidative pathways from catabolic pathways in a host organism. In one embodiment, the altered nucleic acid or polynucleotide lacks a coding for expression of phosphorylation of the product of interest, thereby inactivating phosphorylase activity.

The ASA intermediate may be further converted to the desired end product, for example ASA or erythorbic acid. To produce the ASA intermediate, any host cell capable of converting a carbon source to DKG may be used. Preferred strains of the Enterobacteriaceae family are strains which produce 2, 5-diketo-D-gluconic acid from D-glucose solutions, including Pantoea, described in Kageyama et al (1992) International Journal of Systematic Bacteriology 42, p 203-210. In a preferred embodiment, the host cell is Pantoea citrea in which part or all of the polynucleotide encoding endogenous glucokinase (encoded by the nucleic acid shown in SEQ ID NO: 1) is deleted and part or all of the polynucleotide encoding endogenous gluconokinase (encoded by the nucleic acid shown in SEQ ID NO: 3) is deleted.

The production of ASA intermediates may be in a fermentation environment, i.e., an in vivo environment, or a non-fermentation environment, i.e., an in vitro environment; or in combination with in vivo/in vitro environments. In the methods described further below, the host cell or in vitro environment additionally comprises a heterologous genome that inactivates the coding segment for phosphorylation activity (e.g., glucokinase sequence and gluconokinase sequence).

A. In vivo biocatalytic environment

The present invention comprises the use of a host cell comprising an alteration in a polynucleotide encoding an endogenous enzymatic activity for the phosphorylation of D-glucose at the 6 th carbon, and/or an alteration in a polynucleotide encoding an enzymatic activity for the phosphorylation of D-gluconate at the 6 th carbon, for the in vivo production of an ASA intermediate. The host cell is cultured in an environment with a suitable carbon source, such as a 6-carbon sugar (e.g., glucose), or a 6-carbon sugar acid, or a combination of 6-carbon sugar and/or 6-carbon sugar acid, typically used by strains of the enterobacteriaceae family, to begin biocatalysis. Other carbon sources include, but are not limited to, galactose, lactose, fructose, or enzymatic derivatives thereof. In addition to a suitable carbon source, the fermentation medium must contain suitable minerals, salts, cofactors, buffers and other components known to those skilled in the art for the growth of the culture and to facilitate the enzymatic pathways required for the production of the desired end product.

In the illustrated in-pantoea pathway, D-glucose is subjected to a series of membrane oxidation steps that may include enzymatic conversions of D-glucose dehydrogenase, D-gluconate dehydrogenase, and 2-keto-D-gluconate dehydrogenase, producing intermediates that may include, but are not limited to, GA, KDG, and DKG, see fig. 1. These intermediates are subjected to an intracellular reduction step which may include a series of enzymatic transformations of 2, 5-diketo-D-gluconate reductase (DKGR), 2-ketoreductase (2-KR) and 5-ketoreductase (5-KR) to yield end products including, but not limited to, KLG and IA. In a preferred embodiment of the in vivo environment for the production of ASA intermediates, 5-KR activity is deleted to avoid consumption of IA. Other embodiments have other nucleic acid fragments of desired inactivation depending on the product of interest and the pathway of interest to be inactivated.

If the intermediate of interest is KLG, a nucleic acid encoding DKGR is recombinantly introduced into a strain of pantoea fermentation. It has been found that many bacterial species comprise DKGR, in particular members of the genus corynebacterium, including corynebacterium, brevibacterium and arthrobacter.

In certain embodiments of the invention, 2, 5-DKGR of the Corynebacterium strain SHS752001 (Gridley et al, 1988, Applied and Environmental Microbiology 54: 1770-1775) is recombinantly introduced into a Pantoea strain. Anderson et al, U.S. Pat. No. 5,008,193, disclose the use of Erwinia herbicola (Erwinia herbicoloa) to produce recombinant 2, 5-DKG reductase. Table I provides additional sources of DKG reductase.

The fermentation can be carried out using a batch process or a continuous process. In the batch process, the entire fermentation broth is collected simultaneously, whatever is added. In a continuous system, the fermentation broth is periodically withdrawn for downstream processing while fresh substrate is added. The resulting intermediates can be recovered from the fermentation broth using a variety of methods, including ion exchange resins, absorption or ion retardation resins, activated carbon, concentration-crystallization, membrane filtration, and the like.

B. In vitro biocatalytic environment

The present invention provides biocatalytic production of ASA intermediates, such as KDG, DKG and KLG, from a carbon source in an in vitro or non-fermentation environment, such as a bioreactor. The cells for growth are first cultured, the carbon source for growth is removed for non-fermentative processes, the pH is maintained at about pH4-9, and oxygen is supplied.

Depending on the intermediate of interest produced, the process may require the presence of an enzymatic cofactor. In preferred embodiments disclosed herein, the enzymatic cofactor is regenerated. In certain embodiments, where KDG is the ASA intermediate of interest produced, the bioreactor comprises a viable or non-viable Pantoea citrea host cell comprising an alteration in a polynucleotide encoding normally endogenous enzymatic activity for phosphorylating D-glucose at the 6 th carbon, and/or an alteration in a polynucleotide encoding enzymatic activity for phosphorylating D-gluconate at the 6 th carbon. In this embodiment, the host cell also has mutations in genes encoding glucokinase (glk) and gluconokinase (gntk) activities. In this embodiment, the biocatalytic conversion of the carbon source to KDG by two oxidation steps reduces the loss of the substrate glucose or gluconic acid to the catabolic pathway. In this embodiment, the yield of the intermediate of interest is increased compared to the wild type.

When DKG is the ASA intermediate of interest produced, the bioreactor is provided with living or non-living pantoea citrea host cells comprising an alteration of the activity of a polynucleotide encoding an endogenous glucokinase and gluconokinase that phosphorylates D-glucose at the 6 th carbon, and/or an alteration of the activity of a polynucleotide encoding an enzyme that phosphorylates D-gluconate at the 6 th carbon, and a carbon source biocatalytically convertible to DKG by three oxidation steps. In this embodiment, cofactor regeneration is not required.

When KLG is an ASA intermediate of interest, the bioreactor is provided with a viable or non-viable Pantoea citreum host cell comprising an alteration of a polynucleotide lacking or not encoding an endogenous enzymatic activity for diverting glucose to a catabolic pathway, i.e. lacking the ability to phosphorylate D-glucose at the 6 th carbon, and/or an altered polynucleotide lacking or not encoding an enzymatic activity for phosphorylating D-gluconate at the 6 th carbon, and a carbon source, e.g. D-glucose, biocatalytically convertible to KLG by three oxidation steps and one reduction step. In this embodiment, the reductase activity may be encoded by a nucleic acid contained by, or provided exogenously from, a Pantoea citrea host cell. In this embodiment, the first oxidase activity requires an oxidized form of the cofactor and the reductase activity requires a reduced form of the cofactor. In a preferred embodiment disclosed herein, the pantoea citrifolia cells are altered to remove naturally occurring GDH activity, and a heterologous GDH activity with NADPH + specificity, such as that obtained from a thermoacidophile (t. acidophilum), Cryptococcus unigatus or bacillus species, is introduced into the pantoea cells to provide the desired cofactor recycling system and regenerate the cofactor. In this embodiment, the host cell further comprises a nucleic acid encoding 2, 5-DKG reductase activity, or the 2, 5-DKG is added exogenously to the bioreactor.

In another embodiment for the preparation of KLG, the bioreactor is charged with Pantoea citrea cells comprising an alteration of a nucleic acid encoding an enzymatic activity endogenous to carbon phosphorylating D-glucose at position 6, and/or an alteration of a nucleic acid encoding an enzymatic activity of carbon phosphorylating D-gluconate at position 6, and additionally comprising a nucleic acid encoding a membrane bound GDH, a suitable enzyme and cofactors, and adding D-gluconate which is convertible to DKG. The reaction mixture was then deoxygenated and glucose was added. GDH converts glucose to GA and reductase converts DKG to KLG, while co-factors are regenerated. When these reactions were complete, oxygen was added to convert GA to DKG and the cycle was continued.

In the in vitro biocatalytic process, the carbon source and its metabolites undergo an enzymatic oxidation step, or enzymatic oxidation and enzymatic reduction steps, which occur outside the intracellular environment of the host cell and utilize the enzymatic activities associated with the host cell, and produce the ASA intermediate of interest via a pathway. The enzymatic steps may be performed sequentially or simultaneously in a bioreactor, some requiring cofactors to produce the ASA intermediates of interest. The present invention comprises an in vitro method wherein a host cell is treated by organic matter to render it non-viable, but still available, enzymes for the oxidation and reduction of a carbon source of interest and/or a metabolite thereof to ASA intermediates in biocatalysis of the carbon source.

The bioreactor can be operated using either a batch process or a continuous process. In a batch system, the entire fermentation broth is collected simultaneously, whatever is added. In a continuous system, the fermentation broth is periodically withdrawn for downstream processing while fresh substrate is added. The resulting intermediates can be recovered from the fermentation broth using a variety of methods, including ion exchange resins, absorption or ion retardation resins, activated carbon, concentration-crystallization, membrane filtration, and the like.

In certain embodiments, the host cell may be permeabilized or lyophilized (Izumi et al, J.Ferment. Technol.61(1983)135-142), as long as the enzymatic activity necessary for the conversion of the carbon source or derivative thereof is still available, while reducing the fraction of the carbon source or derivative thereof diverted to the catabolic pathway. Bioreactors can take advantage of certain enzyme activities provided from external sources, and operate in environments that provide solvents or long polymers that stabilize or enhance enzyme activities.

C. Host cell producing ASA

If any oxidation or reduction enzyme that is required to direct the conversion of the host cell carbohydrate pathway to an ASA intermediate (such as KDG, DKG or KLG) does not naturally occur in the host cell, it can be introduced by recombinant DNA techniques known to those skilled in the art. In addition, enzymes that interfere with unwanted catabolic pathways can be inactivated by recombinant DNA methods. The present invention encompasses the recombinant introduction or inactivation of any enzyme or intermediate required to achieve the desired pathway.

In certain embodiments, ASA is produced using an enterobacteriaceae strain corrected by a recessive plasmid, see PCT WO 98/59054.

In certain embodiments, the host cell used to produce the ASA intermediate is pantoea citrea, e.g., ATCC accession No. 39140. Nucleic acids encoding oxidizing or reducing enzymes useful for the production of ASA intermediates in Pantoea species include the following sources:

TABLE I

Enzyme Reference to

Glucose dehydrogenase Smith et al 1989, Biochem.

J.261: 973 of the total weight of the composition; neijssel et al

1989，Antonie Van

Leauvenhoek 56(1)：51-61

Gluconate dehydrogenase Matsushita et al 1979,

J.Biochem.85：1173；Kulbe

et al 1987, Ann.N.Y.Acad

Sci 6：552

2-keto-D-gluconate dehydrogenase Stroshane1977

Biotechnol.BioEng9(4)

459

2-ketogluconate reductase J.Gen.Microbiol.1991,

137：1479

2, 5-diketo-D-gluconate reductase U.S. Pat. Nos.: 5,795,761, respectively;

5,376,544；5,583,025；

4,757,012；4,758,514；

5,008,193；5,004,690；

5,032,514

recovery of ASA intermediates

Once produced, the ASA intermediate may be recovered and/or purified using any method known to those skilled in the art, including freeze drying, crystallization, spray drying, electrodialysis, and the like. For example, U.S. patent 5747306 issued 5/1998 and 4767870 issued 8/30/1998 describe electrodialysis methods for purifying ASA and ASA intermediates such as KLG. Alternatively, the intermediate may be formulated directly from the fermentation broth or bioreactor and granulated or added to the liquor.

KLG produced by the methods of the present invention can be further converted to ascorbic acid and KDG to erythorbic acid using methods known to those skilled in the art, see, for example, Reichstein and Grussner, helv, chim. acta, 17, 311-328 (1934). Ascorbic acid may have 4 stereoisomers: l-ascorbic acid, D-arabino-ascorbic acid (erythorbic acid), L-arabino-ascorbic acid and D-xylo-ascorbic acid exhibiting vitamin C activity.

E. Conditions of analysis

Methods for detecting ASA intermediates, ASA and ASA stereoisomers include redox titration using 2, 6-dichloroindophenol (Burton et al 1979, j.assoc. pub. analyts17: 105) or other suitable reagents; high Performance Liquid Chromatography (HPLC) using anion exchange (J.Chrom.1980, 196: 163); and electro-redox processes (Pachia, 1976, anal. chem.48: 364). The skilled artisan is well aware of the controls used in using these detection methods.

Fermentation medium:

the fermentation medium of the present invention must contain suitable carbon substrates including, but not limited to, monosaccharides such as glucose, oligosaccharides such as lactose or sucrose, polysaccharides such as starch or cellulose, and unpurified mixtures from renewable feedstocks such as cheese whey permeate (cheese whey), corn steep liquor (corn sugar beet syrup), and barley malt. Alternatively, the carbon substrate may be a single carbon substrate, such as carbon. Although it is contemplated that the carbon source used in the present invention may comprise a variety of carbon-containing substrates and is limited only by the organism selected, preferred carbon substrates include glucose and/or fructose and mixtures thereof. The use of a mixture of glucose and fructose, together with the altered genome described herein, to separate the oxidative and catabolic pathways, allows for the utilization of fructose to meet the metabolic needs of the host cell by utilizing glucose to increase yield and conversion to the ascorbic acid intermediate of interest.

While all of the above carbon substrates are contemplated as being suitable for the present invention, the carbohydrates glucose, fructose or sucrose are preferred. The carbon substrate concentration is about 55% to about 75% w/w. Preferably, the concentration is about 60-70% w/w. The inventors most preferably used 60% or 67% glucose.

In addition to a suitable carbon source, the fermentation medium must contain suitable minerals, salts, vitamins, cofactors and buffers suitable for growth or culture and to facilitate the enzymatic pathways required for the production of ascorbic acid intermediates.

The culture conditions are as follows:

preculture:

cell cultures are generally grown in a suitable medium at 25-32 deg.C, preferably about 28 or 29 deg.C. Although the examples describe the growth medium used, other typical growth media for use in the present invention are common commercially prepared media, such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth, or Yeast Medium (YM) broth. Other defined, or synthetic, growth media may also be used, and certain persons skilled in the art of microbiology or fermentation science will know the appropriate medium for growth of a particular microorganism.

Preferably, a suitable pH range for fermentation is pH5-8, a seed bottle pH7-7.5, and a reaction vessel pH 5-6.

It will be appreciated by those skilled in the art of fermentative microbiology that since applicants have demonstrated the feasibility of the process of the present invention, many factors affecting the fermentation process may have to be optimized and controlled to maximize the production of ascorbic acid intermediates. Many of these factors, such as pH, carbon source concentration, and dissolved oxygen levels, may affect enzymatic processes depending on the cell type used to produce the ascorbic acid intermediate.

Batch and continuous fermentation

The culture system of the present process uses a fed-batch fermentation process. Traditional batch fermentations are closed systems with the medium composition set at the beginning of the fermentation and not subject to manual changes during the fermentation. Thus, the medium is inoculated with one or more organisms of interest at the start of the fermentation, which does not require any addition of substances to the system. However, "batch" fermentation is generally a batch addition of carbon source and often seeks to control factors such as pH and oxygen concentration. In batch systems, the metabolite and biomass composition of the system often changes until fermentation is terminated. In batch culture, cells reach a stationary lag phase steadily to a high growth log phase, and finally to a stationary phase where the growth rate is reduced or stopped. If left untreated, the quiescent cells will eventually die. Logarithmic phase cells are generally associated with a large production of end products or intermediates.

The Fed-Batch system is a variant of the standard Batch system. The present invention is also applicable to Fed-Batch fermentation processes which comprise a typical Batch system with the addition of substrate in increments during the fermentation process. The Fed-Batch system is used when catabolite repression tends to inhibit cellular metabolism and limited amounts of substrate in the culture medium are desired. It is difficult to determine the actual substrate concentration of Fed-Batch and thus depends on changes in measurable factors, such as pH, dissolved oxygen, and exhaust gases such as CO₂The partial pressure of (c) is estimated. Batch and Fed-Batch fermentations are common and well known in the art, examples are found in Brock, supra.

Although the present invention is carried out in a batch mode, it is contemplated that the process should be suitable for a continuous fermentation process. Continuous fermentation is an open system in which a defined fermentation medium is continuously added to a bioreactor while an equal amount of conditioned medium is removed for processing. Continuous fermentation generally maintains a culture at a constant high density, with cells mainly in log phase growth.

Continuous fermentation can modulate one or more factors that affect cell growth or final product concentration. For example, one method maintains a restrictive nutrient (e.g., carbon source or nitrogen level) fixation and adjusts all other parameters. In other systems, many factors affecting growth can be continuously varied while keeping the cell concentration constant as measured by media turbidity. Continuous systems strive to maintain steady state growth conditions, and therefore cell loss due to medium withdrawal must be balanced with the growth rate of the cells in the fermentation. Methods for modulating nutrients and growth factors for continuous fermentation processes and techniques for maximizing product formation rates are well known in the art of industrial microbiology and various methods are detailed by Brock (supra).

It is contemplated that the present invention may be practiced using batch, fed-batch or continuous processes and suitable known fermentation practices. In addition, it is contemplated that cells may be immobilized on a substrate as a monolithic cell catalyst for the production of ascorbic acid intermediates under fermentation conditions.

Identification and purification of ascorbic acid intermediates:

methods for purifying ascorbic acid intermediates of interest from fermentation media are known in the art.

The media can be analyzed by High Pressure Liquid Chromatography (HPLC) to directly identify specific ascorbic acid intermediates. The preferred method of the invention uses an analytical ion exchange column with a mobile phase of 0.01N sulphuric acid to analyse the fermentation medium in an equivalent manner.

Examples

General procedure

Materials and methods suitable for maintaining and growing bacterial cultures are described in Manual of methods for General Bacteriology (phillip Gerhardt, r.g.e.murray, Ralph n.costilow, Eugene w.nester, Willis a.wood, noil r.krieg and g.briggs Phillips), p.210-: a Textbook of Industrial microbiology, second edition (1989) Sinauer Associates, Inc., Sunderland, Mass. Unless otherwise indicated, all reagents and materials used for bacterial cell growth were obtained from Diffco Laboratories (Detroit, Mich.), Aldrich Chemicals (Milwaukee, Wis.) or Sigma Chemical Company (St. Louis, Mo.).

Growth media for the preculture or inoculum are commercially available, and preparations such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth or Yeast Medium (YM) broth are obtained from GIBCO/BRL (Gaithersburg, Md.). LB-50amp is Luria-Bertani broth containing 50. mu.g/ml ampicillin.

Fermentation medium:

two basic fermentation media were prepared for use in the examples below, designated seed bottle medium and fermentation medium. These minimal media are altered by changing the carbon source or adding other agents (e.g., sulfite). The reagent for each culture medium includes KH₂PO₄、K₂HPO₄、MgSO₄·7H₂O, Difco Soytone, sodium citrate, fructose, (NH)₄)₂SO₄Nicotinic acid, FeCl₃·6H₂O and trace salts including but not limited to ZnSO₄·7H₂O、MnSO₄·H₂O and Na₂MoO₄·2H₂O)；KH₂PO₄、MgSO₄·7H₂O、(NH₄)₂SO₄Monosodium glutamate, ZnSO₄·7H₂O、MnSO₄·H₂O、Na₂MoO₄·2H₂O、FeCl₃·6H₂O, choline chloride, MazuDF-204 (antifoam), nicotinic acid, calcium pantothenate and HFCS (42 DE). HFCS may also be prepared in the desired ratio of glucose to fructose, for example a fructose/glucose solution consisting of 27.3g/L fructose powder, 25.0g/L glucose powder.

Cell:

all commercial cells used in the following examples were obtained from ATCC and identified herein by their ATCC number. Recombinant Pantoea citrea cells (ATCC39140) were used as ascorbic acid intermediate-producing cells, and constructed as described in examples 4 and 5. Enzymatic and genomic analysis revealed that the strains MDP41 and DD6 lack the genes encoding glucokinase, gluconokinase and both enzymes, whereas the wild-type strain contains the genes encoding glucokinase and/or gluconokinase.

Ascorbic acid intermediate analysis:

HPLC analysis was performed to confirm the presence of an ascorbic acid intermediate such as 2-KLG. The fermentation reactor sample was taken from the tank and loaded onto a Dionex (Sunnyvale, Calif., product No. 043118) Ion Pac AS 10 column (4 mm. times.250 mm) connected to a Waters 2690 separation Module and a Waters 410 differential refractometer (Milford, MA).

Method for analyzing ascorbic acid intermediate production

Definitions the methods for determining yield, OUR and CER have been described previously.

Recombination method

Vector sequences

The expression vector used in the method of the invention comprises at least one promoter associated with the enzyme, which promoter is functional in the host cell. In one embodiment of the invention, the promoter is the wild-type promoter for the selected enzyme, and in another embodiment of the invention, the promoter is heterologous to the enzyme but still functional in the host cell. In one embodiment of the invention, the nucleic acid encoding the enzyme is stably integrated into the genome of the microorganism.

In certain embodiments, the expression vector comprises a multiple cloning site cassette, preferably comprising at least one restriction endonuclease site unique to the vector to facilitate nucleic acid manipulation. In a preferred embodiment, the vector further comprises one or more selectable markers. The term selectable marker as used herein refers to a gene capable of being expressed in a host microorganism, allowing for easy selection of a host comprising the vector. Examples of such selectable markers include, but are not limited to, antibiotics such as erythromycin, actinomycin, chloramphenicol, and tetracycline.

A preferred plasmid for The recombinant introduction of non-naturally occurring enzymes or intermediates into strains of The Enterobacteriaceae family is a transmissible, but non-self transmissible plasmid RSF1010 which has The ability to replicate in a variety of bacterial hosts, including Gram-and Gram + bacteria (Frey et al, 1989, TheMolecular biology of IncQ plasmids in: the material of the material is produced by Thomas (eds),Promiscuous Plasmids of Gram Negative Bacteriaacademycpress, London, pages 79-94). Frey et al (1992, Gene 113: 101-106) reported that 3 regions were found to affect the transfer properties of RSF 1010.

Transformation of

Current Protocols In Molecular Biology (Vol 1, Ausubel et al, John Wiley&Sons, Inc.1987, Chapter 9) teaches general transformation methods, including calcium phosphate methods, transformation using DEAE-dextran, and electroporation. Various transformation methods are known to those skilled in the art for introducing a nucleic acid encoding a protein of interest into a given host cell. A variety of host cells can be used to recombinantly produce exogenously added pathway enzymes, including bacterial, fungal, mammalian, insect, and plant cells.

In certain embodiments of this method, the host cell is an enterobacteriaceae cell. Enterobacteriaceae includes Erwinia, Enterobacter, Gluconobacter and Pantoea species. The preferred Enterobacteriaceae fermenting strains used according to the invention for producing ASA intermediates are Pantoea species, in particular Pantoea citrea. In certain embodiments, the host cell is pantoea citrea comprising pathway enzymes capable of converting the carbon source to KLG.

Identification of transformants

Whether the transformed host cell can be detected by the presence/absence of expression of the marker gene, it is recommended that the expression of the nucleic acid of interest should be confirmed regardless of the presence or absence thereof. For example, if a nucleic acid encoding a pathway enzyme is inserted within the sequence of a marker gene, a recombinant cell comprising the insertion can be identified by the lack of a loss of function of the marker gene. Alternatively, a marker gene may be placed in tandem with a nucleic acid encoding a pathway enzyme under the control of a single promoter. Marker genes are expressed in response to induction or selection, usually indicating that the enzyme is also expressed.

In addition, host cells comprising pathway enzyme coding sequences and expressing the enzymes can be identified using a variety of methods known to those skilled in the art. These methods include, but are not limited to, DNA-DNA or DNA-RNA hybridization and protein biological analysis or immunoassay techniques, including membrane, solution or chip based techniques, for detecting and/or quantifying nucleic acids or proteins.

In addition, the presence of the polynucleotide sequence of the enzyme in the host microorganism can be detected by DNA-DNA or DNA-RNA hybridization or amplification using the probe, a portion or fragment of the polynucleotide sequence of the enzyme.

The manner and method of carrying out the invention will be more fully understood by those skilled in the art with reference to the following examples, which are not intended to limit the scope of the invention or its claims in any way. All references and patent publications referred to herein are incorporated herein by reference.

Examples

Example 1

Construction of Pantoea citrea 139-2a genomic library

Pantoea citrea genomic DNA was prepared using a DNA-Pure TM genomic DNA isolation kit (CPG, Lincoln Park, NJ). 50. mu.g of DNA was partially digested with the restriction enzyme Sau3A according to the manufacturer's instructions (Roche molecular biochemicals, Indianapolis, IN). The digestion products were separated using a 1% agarose gel and 3-5kb DNA fragments were purified from the gel using the Qiaquick gel extraction kit (Qiagen Inc. Valencia, Calif.). The resulting DNA was ligated with the BamHI linearized plasmid pBK-CMV (Stratagene, La Jolla, Calif.). A library of about 10xx different plasmids was thus obtained.

Example 2

Isolation of structural Gene of glucokinase

To select plasmids carrying the glucokinase gene from Pantoea citrea, the genomic library (see above) was transformed into the E.coli strain NF9, glk lacking the glucokinase gene (glkA) and the PTS transport system^-(Floreset al, nat. Biotech.14, 620-. After transformation, cells grown on glucose as the sole carbon source in M9 medium were selected. Using this strategy to select the candidate with glk^-Or pts^-Mutation of the complementary plasmid.

After 48 hours incubation at 37 ℃ many clones appeared. Partial clones were further purified, plasmids isolated, and their characteristics were restriction analyzed. All plasmids were found to contain common DNA fragments.

These plasmids were retransformed back to NF9, glk^-All of them can grow in M9-glucose medium, and they were confirmed to complement NF9, glk^-At least one mutation present in (a).

The plasmid pMD4, which contained an insert of approximately 3.9kb, was thus isolated. The insert of this plasmid was sequenced and found to have a gene with strong similarity to the E.coli glkA gene in a region of about 1010 bp. (SEQ ID 4.)

Example 3

Inactivation of the glucokinase gene by homologous recombination.

The general strategy for inactivation of genes by homologous recombination using a suicide vector has been described previously (Miller and Mekalanos, J.Bacteriol.170(1988) 2575-2583). To inactivate the glk gene of Pantoea citrea using this method, two plasmids were constructed: pMD5 and pMD 6.

To construct pMD5, plasmid pMD4 was digested with NcoI and SnaBI restriction enzymes according to the manufacturer's instructions. (Roche Molecular Biochemicals, Indianapolis, IN). The cohesive ends generated by the above enzymes were blunted using T4 polymerase using standard techniques. This DNA was ligated with a loxP-Cat-loxP cassette isolated from pLoxCat2 SpeI-EcoRV DNA fragment. (Palmerios et al, Gene (2000)247, 255-264.). The cassette encodes for chloramphenicol resistance. The ligation mixture was transformed into TOP10 competent cells (Invitrogen, Carlsbard CA) and selected for growth in 10. mu.g/ml chloramphenicol. Several clones were obtained after 18 hours incubation at 37 ℃. Plasmids from certain clones were purified and characterized by restriction analysis. The presence of loxP-Cat-loxP was confirmed, as well as the deletion of the DNA region between the NcoI and SnaBI sites of the glk gene. A plasmid with these properties was designated pMD 5.

To construct pMD6, plasmid pMD5 was digested with BamHI and CelII restriction enzymes. The DNA fragment containing the glk gene interrupted by the loxP-cassette was ligated with the EcoRV-BsaI DNA fragment isolated from plasmid pR6Koril (unpublished results). This fragment contains the R6K origin of replication and the kanamycin resistance gene. The mixture was ligated to transformation of strain SY327(Miller and Mekalanos, supra) and transformants were selected on plates containing kanamycin and chloramphenicol (20 and 10. mu.g/ml, respectively). Several clones were obtained after incubation at 37 ℃ for 24 hours. Plasmids from certain clones were purified and characterized by restriction analysis. Confirming the presence of loxP-Cat-loxP and the R6K starting point. The plasmid with these characteristics was designated pMD 6.

In general, one of the characteristics of the pMD6 and R6K derivatives is that they can only replicate in strains carrying the pir gene of the plasmid R6K (Miller and mekalanos, supra). Pantoea citrea does not contain the pir gene or maintain replication of pMD 6. After transformation of pMD6 into Pantoea citrea 139-2a and selection of the Cm (R) strain, correct gene replacement was obtained by homologous recombination. Inactivation of the glucokinase gene was confirmed by assaying glucokinase activity using a glucokinase-glucose-6-phosphate dehydrogenase coupled assay as described by Fukuda et al (Fukuda Y., Yamaguchi S., Shimosaka M., Murata K., and Kimura A.J.Bacteriol. (1983) Vol 156: 922-925). The Pantoea citrea strain, which was confirmed to be glucokinase inactive, was designated MDP 4. The generation of the inactivated glucokinase gene was further confirmed by comparing the size of the PCR product obtained using the chromosomal DNA of the 139-2a or MDP4 strain and primers (SEQ. ID.8, SEQ. ID.9) hybridizing to the glucokinase structural gene. According to this method, the size of the PCR product should reflect that the loxP-Cat-loxP cassette has been cloned into the glucokinase structural gene.

Example 4

Removal of the chloramphenicol resistance marker of MDP4

After overnight growth in YENB medium (0.75% yeast extract, 0.8% nutrient broth) at 30 ℃, an aqueous suspension of Pantoea citrea MDP40 was electroporated using plasmid pJW168 (Palmers et al, Gene (2000)247, 255-. After growth at 30 ℃ in SOC medium, transformants were selected using LB agar medium supplemented with carbenicillin (200. mu.g/ml) and IPTG (1mM) at 30 ℃ (permissive temperature for replication of pJW 168). Mixed clones were transferred twice overnight at 35 ℃ using fresh LB agar medium supplemented with carbenicillin and IPTG to remove the chromosomal chloramphenicol resistance gene by recombination at loxP sites mediated by Cre recombinase (Hoess and Abremski, J.mol.biol., 181: 351-362). The resulting clones were replicated (repleniced) in whole plates onto LB agar medium supplemented with carbenicillin and IPTG and LB agar supplemented with chloramphenicol (12.5. mu.g/ml) to facilitate identification of carbenicillin-resistant, chloramphenicol-sensitive (indicating removal of the marker gene) clones at 30 ℃. 10ml of LB medium were inoculated with an overnight culture at 30 ℃ of one of the above clones. Cultures were incubated overnight at 35 ℃ when grown to an OD (600nm) of 0.6 at 30 ℃. Several dilutions were plated on pre-warmed LB agar medium and plates were incubated overnight at 35 ℃ (an unlicensed temperature replicated by pJW 168). The resulting clones were replicated in whole plates to LB agar medium and LB agar medium supplemented with carbenicillin (200. mu.g/ml) to identify clones sensitive to carbenicillin (indicating loss of plasmid pJW168) at 30 ℃. Primers SEQ ID NO: 5 and SEQ ID NO: 6, a further analysis of the glK mutant MDP41 described above by genomic PCR yielded the expected PCR product (data not shown).

Example 5

Inactivation of the gluconokinase gene by homologous recombination.

The general strategy for inactivating the gluconokinase gene of Pantoea citrea is shown in FIG. 9, and is substantially the same as the strategy for inactivating the glucokinase gene described in example 3. Briefly, after isolation and sequencing of the plasmid that allowed growth of e.coli strain □ gntK □ idnK using gluconic acid as the sole carbon source (data not shown), primers of seq.id NO: 10 and seq id no: 11, a DNA fragment containing a structural gene of the gluconokinase gene was produced by PCR. The PCR product of about 3kb was cloned into a multicopy plasmid containing the R6K origin of replication. Using a nucleic acid sequence located in seq id NO: 2 into the loxP-Cat-loxP cassette is inserted the only PstI restriction site of the gluconokinase structural gene. This construct was transferred into the chromosome of Pantoea citrea strain MDP41 by homologous recombination. Primers SEQ ID NO: 11 and SEQ ID NO: 12, confirmation of accurate disruption of gluconokinase by the loxP-Cat-loxP cassette by PCR.

The new strain with both glucose and gluconokinase inactivated was named MDP 5. This strain still contains the Cat marker inserted in the gluconokinase structural gene. By repeating the procedure described in example 4, a marker-free strain was obtained, which was designated DD 6.

Example 6

It is elucidated hereinafter that double-deleted host cells (pantoea host cells deficient in glucokinase and gluconokinase) require O₂And (4) the advantages of the aspects.

Seed strain:

one tube of the culture stored in liquid nitrogen was dissolved in air and 0.75mL was added to a 2-L sterile Erlenmeyer flask containing 500mL of seed medium. The Erlenmeyer flask was incubated at 29 ℃ at 250 rpm for 12 hours. The switching standard is OD₅₅₀Greater than 2.5.

Seed bottle culture medium

The medium composition was prepared as follows:

amount of ingredient

KH₂PO₄12.0g/L

K₂HPO₄4.0g/L

MgSO₄·7H₂O 2.0g/L

Difco Soytone 2.0g/L

Sodium citrate 0.1g/L

Fructose 5.0g/L

(NH₄)₂SO₄1.0g/L

Nicotinic acid 0.02g/L

FeCl₃·6H₂O 5mLL (0.4g/L stock solution)

5mL/L of trace salt (following solution: 0.58g/L ZnSO)₄·7H₂O，0.34

g/L MnSO₄·H₂O，0.48g/L Na₂MoO₄·2H₂O)

The pH of the medium solution was adjusted to 7.0. + -. 0.1 units using 20% NaOH. Tetracycline hydrochloride was added to a final concentration of 20mg/L (2mL/L of a 10g/L stock solution). The resulting culture medium solution was filter sterilized using a 0.2 μ filter apparatus. The medium was then autoclaved and 500mL of autoclaved medium was added to a 2-L Erlenmeyer flask.

Production of fermentation tank

Additives before sterilization of reaction vessels

Concentration of ingredients

KH₂PO₄3.5g/L

MgSO₄·7H₂O 1.0g/L

(NH₄)₂SO₄0.92g/L

Monosodium glutamate 15.0g/L

ZnSO₄·7H₂O 5.79mg/L

MnSO₄·H₂O 3.44mg/L

Na₂MoO₄·2H₂O 4.70mg/L

FeCl₃·6H₂O 2.20mg/L

Choline chloride 0.112g/L

Mazu DF-204 0.167g/L

The culture medium constructed as described above was sterilized at 121 ℃ for 45 minutes.

After the tank had been sterilized, the following additives were added to the fermentor:

concentration of ingredients

Nicotinic acid 16.8mg/L

Calcium pantothenate 3.36mg/L

HFCS (42DE) 95.5g/L (gluconic acid or glucose as specific starting substrate if desired)

After sterilization and addition of the sterilized ingredients, the final volume was 6.0L. The entire seed bottle contents prepared as described were inoculated in the thus prepared tank and medium, resulting in a volume of 6.5L.

The growth conditions were 29 ℃ and pH 6.0. The stirring rate, back pressure and air flow were adjusted as needed to maintain dissolved oxygen above zero.

Results

The oxidation pathway of the ascorbic acid intermediate is depicted in figure 10. Since CO is present₂The only carbon source of (A) is from the carbon substrate, no additional CO is supplied to the reaction vessel₂Thus, by determining carbon dioxide production (CER), the number of carbons utilized by the catabolic pathway can be calculated, thereby determining the uncoupling of the catabolic and production (oxidation) pathways. By CER measurements, 63% of the glucose was converted to ascorbic acid intermediates and 37% to catabolites when the wild-type organism was used during fermentation (figure 12). In the second phase of the study, the nucleic acid encoding glucokinase was expressed under wild-type conditions. CO determination by CER as shown in FIG. 13A₂Is reduced to about 18%. Glucose catabolism is thus reduced but not completely uncoupled. In an attempt to determine the source, i.e., the pathway by which the carbon substrate is diverted to the catabolic pathway, gluconic acid is provided as the sole carbon source. As shown by comparison of FIG. 13B with FIG. 13A, gluconic acid is catabolized at about the same rate as glucose as the carbon substrate. (83% conversion of gluconic acid to ascorbic acid intermediate, 17% diversion of gluconic acid to the catabolic pathway (determined by CER)). See also table 2:

TABLE 2

Bacterial strains	Conversion of the glucose component to Metabolic DKG		Conversion of gluconic acid component to Metabolic DKG
					Wild type	0.37	0.63	--	--
Glucokinase deficiency (glkA)	0.18	0.82	0.17	0.83
					Glucokinase deletion (gntK)	0.24	0.76	0.02	0.98

The final phase of the study was performed by examining OUR and CER for host cells that had been deleted in their genome and encoded by the glucokinase and gluconokinase genomes. As shown in figure 14 of the drawings,conversion of 3% glucose to CO₂While the control (wild type) showed 43% glucose production of CO₂. Thus, the wild type shows higher glucose catabolism through catabolic pathways, resulting in reduced yields and high oxygen requirements. However, the double deletion of glucokinase and gluconokinase inactivates catabolism to less than 10%, less than 5%, and particularly less than 3% or less of the original carbon substrate.

Conclusion

The double mutation of glucokinase and gluconokinase shunts almost all glucose (about 98%) to 2, 5-DKG.

Example 7

Glycerol is produced from fructose.

To demonstrate that pantoea citrea can be used to produce compounds derived from fructose, empage et al [ expression, m., Haynie, s., laffund, l., Pucci, j. and rated, g.process for the biological production of 1, 3-general with high patent: WO 0112833-A412001, month 2 and day 22; e.i. du PONT DE NEMOURS AND COMPANY; glycerol was produced by the method described in GENENCOR INTERNATIONAL, INC. Briefly, this method uses two enzymes of yeast to convert dihydroxyacetone phosphate (DHAP) to glycerol as shown in the reaction depicted in fig. 25.

The genes for the GPD1 and GPP2 enzymes were cloned into the multicopy plasmid pTrc99 under the control of the Trc promoter (Empage et al, 2001). This plasmid (pAH48) was able to produce high levels of both enzymes.

The present inventors considered that, in order to produce glycerol in Pantoea citrea, it is desirable to eliminate or reduce the natural ability of the strain to assimilate glycerol. The glycerol catabolic pathway common in many bacteria is through the action of glycerol kinases [ Lin e.c.ann.rev.microbiol.1976.30: 535-578.Glycerol dispersion and its regulation in bacteria]. The present inventors found that Pantoea citrea was able to grow in a medium containing glycerol as the sole carbon source. In addition, the genomic sequence of Pantoea citrea was examined, indicating that it has a glycerol kinase gene very similar to the glkA gene of E.coli.

Therefore, the structural gene (glpK gene) of the glycerol kinase is inactivated in order to eliminate the activity. This was achieved as described in examples 3 and 5 (inactivation of the glucokinase and gluconokinase genes). Briefly, a 2.9kb DNA fragment comprising the glpK gene and flanking sequences was obtained by PCR using pantoea citriodora chromosomal DNA and the primers disclosed in SEQ id. glpK1 and SEQ id. glpK 2. This 2.9kb DNA fragment was cloned into the R6K vector as shown in examples 3 and 5. The DNA sequence of glpK gene is shown in SEQ ID. The DNA sequence of glpK was checked to show the presence of HpaI site and was selected for the insertion of loxP-Cat-loxP cassette. Once the plasmid construct of interest was obtained, the glpK disruption was transferred into the chromosome of Pantoea citrea strain 139-2a ps-by homologous recombination as described in examples 3 and 5. The resulting Pantoea citrea glpK: Cm strain was designated MDG 1.

Once the presence of interruption of the glpK gene in the genome of Pantoea citrea was confirmed, the effect of the mutation was evaluated. For this purpose, strain MDG1 was grown in minimal medium M9 containing 0.4% glycerol as sole carbon source. After 48 hours incubation of the cells at 30 ℃, no growthwas observed, indicating that strain MDG1 lost the ability to utilize glycerol as a carbon source.

The strain MDG1 was transformed with the plasmid pAH48(Emptage et al, 2001), and the resulting strain MDG2 was tested for its ability to produce glycerol using fructose as the sole carbon source. The test was performed by incubating the strain in minimal medium containing 2% fructose as sole carbon source. After 24 hours incubation of the cells at 30 ℃, samples were collected for HPLC analysis as described by Emptage et al (2001). It was thus found that strain MDG1 did not produce any glycerol, whereas strain MDG2 produced 1.36g/L glycerol. These results demonstrate that Pantoea citrea is able to convert most of fructose to form glycerol.

Other various examples and modifications of the foregoing description and embodiments will be apparent to those skilled in the art upon reading this disclosure without departing from the spirit and scope of the invention, and it is intended that all such examples and modifications be included within the scope of the appended claims. All publications and patents referred to herein are incorporated by reference in their entirety.

Claims (12)

1. A method for producing an ascorbic acid intermediate in a recombinant host cell comprising culturing a host cell capable of producing said ascorbic acid intermediate in the presence of glucose under conditions suitable for the production of said ascorbic acid intermediate, wherein said host cell comprises a deletion of at least one polynucleotide encoding a kinase selected from the group consisting of glucokinase and gluconokinase.

2. The method of claim 1, wherein said host cell further comprises two deletions of coding residues of a kinase selected from the group consisting of glucokinase and gluconokinase.

3. The process of claim 1, further comprising the product recovery step.

4. The method of claim 1, further comprising the step of converting the ascorbic acid intermediate to another product.

5. A method of enhancing the production of an ascorbic acid intermediate from a carbon source comprising,

d) obtaining an altered Pantoea strain comprising an inactivated chromosomal gene in a bacterial host strain,

e) culturing the altered bacterial host strain under suitable conditions, and

f) producing an ascorbic acid intermediate from a carbon source, wherein the production of the ascorbic acid intermediate is enhanced as compared to the production of the ascorbic acid intermediate in an unaltered bacterial host strain.

6. The process of claim 5, wherein the ascorbic acid intermediate is selected from the group consisting of gluconic acid, 2-keto-D-gluconic acid, 2, 5-diketo-D-gluconic acid, 2-keto-L-gulonic acid, L-iduronic acid, erythorbic acid, and tartaric acid.

7. The process of claim 6, wherein the ascorbic acid intermediate is 2, 5-diketo-D-gluconic acid.

8. The method of claim 5, wherein the altered bacterial strain is obtained by inactivating the glk chromosomal gene.

9. The method of claim 5, wherein the altered bacterial strain is obtained by inactivating a gntk chromosomal gene.

10. The method of claim 5, wherein the altered bacterial strain is obtained by inactivating the glk and gntk chromosomal genes.

11. The method of claim 5, wherein said bacterial strain is pantoea.

12. The method of claim 11, wherein said bacterial strain is pantoea citrifolia.

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